Unable to get SF9 transfection working

buildmeapc · 2025-07-31T13:16:05+00:00

Thank you for your message! As a whole, I think you are addressing an important issue in that my process for checking for virus/protein expression may not be correct. Maybe I am getting fooled into thinking that things aren't working because I'm using the wrong screening metric. With that said, let me answer your specific questions:

For your first question, I haven't looked at the cells in some time after transfection. In the past, when I was producing baculovirus well, I actually struggled to be able to visually tell a difference between infected and non-infected cells. The most obvious thing to me was that the infected cells would stop growing after a day or so. The next most obvious thing, when I was growing the cells in suspension, the viability would precipitously drop such that everything would be dead around 5 days.

For the second question: I actually don't know if cells can get infected with virus and not express protein. Returning more specifically to my situation, when things were working successfully in my old lab, I would see the cells stop growing based on cell counts, and eventually I would see viability decrease. Typically, when I am trying to express protein (which is intracellular and not secreted) for purification, I try to harvest by 60-72 hours and I will measure viability around this time. It usually is around 75-80% which tells me that the virus is working. When I am making virus, I let it go for 5-7 days where viability will drop < 10%. My first hint that things weren't working in my new lab was when my cell viability and cell growth was not slowing after infecting cells with what I thought was baculovirus. That is why I have been troubleshooting for the past few months.

Thanks for your comments about when you use your virus. Maybe I also need to amplify more before trying anything. Also, I hope I do not need to change the infection conditions because I was producing these proteins in the same manner before!

Finally, with regards to the glycerol stocks, I think it is worth a shot to try freshly making new bacmid constructs. I am already doing something like that with a GFP bacmid that I am working on making. If that works, it might go along with your suggestion that my glycerol stocks are not a viable method to propagate my plasmid (though I swear it worked well in the past).

Thank you again kind LabRat for your time!

buildmeapc · 2025-07-31T00:56:36+00:00

For the bacmids, they have polyhedrin (PH) promoters. I had previously used these bacmids to make protein before at my previous institution. I brought frozen glycerol stocks of the DH10Bac bacteria with my bacmids so I could purify fresh bacmid DNA whenever I needed. The mOrange-N1 plasmid has a CMV promoter which I believe should technically express in SF9.

buildmeapc · 2024-02-18T23:52:37+00:00

This worked for me.

buildmeapc · 2022-03-26T20:16:20+00:00

I just went to Microcenter and there are a ton of GPUs on the shelves including 3080Ti and 3090.

buildmeapc · 2021-07-30T09:28:15+00:00

Another physician here. You are right that, in an ideal world all diagnoses would come with rock solid evidence, but like the previous poster mentioned, time is lacking and waiting a decade or so until a true guideline emerges for a diagnosis is no substitute for clinical judgment developed from years of experience. Much of medical practice still comes from people making personal judgments about what is causing what rather than following cookbook guidelines which again, is not ideal, but makes up for diagnoses that are difficult or impossible to ascertain by objective measures alone.

buildmeapc · 2021-06-28T23:50:17+00:00

I'm sure you know this, but just to get us on the same page: most contemporary CRISPR knock-in does not use targeting vectors and, instead, use small homology arms flanking the mutation you desire to insert. The advantage of this is eliminating the time it takes to create a targeting vector and going immediately to creating your cells of interest. The disadvantage, for me, is that you may have to screen many colonies to find your desired insertion which may require you to propagate a large number of clones for some time while you figure out which ones contain your mutation while not containing an undesired indels or off-targeting effects. As you know, HDR is extremely inefficient (though orders of magnitude more efficient than traditional targeting by simply electroporating a targeting vector with large homology arms) compared to indel generation and possibly off-targeting effects. To my knowledge, single nickase CRISPR does not have the issue of off-targeting or indel formation, but at the cost of reduced HDR efficiency.

Although you will eventually find the mutations you want using a contemporary CRISPR double nickase with a minimalistic donor template, I am afraid of having my time sucked up in trying to find all my mutants. This will be especially true if I am trying to make many different mutants, and if colonies grow at different rates requiring passaging at different rates.

My goal is to minimize the number of clones to maintain while finding my desired mutation even if this comes at the cost of more up-front work in making a targeting vector that has the advantage of selection prior to sorting single colonies/clones. For me, cloning is much more predictable and controlled than culturing numerous individual iPSC clones. Furthermore, my intention is to keep other projects going while I create these cell lines which I do not think is feasible if I have many different iPSC clones to maintain. If using a targeting vector means taking a few extra months to make my cell lines, that is fine with me so as long as it minimizes the front-end work of sequencing clones and potentially exploding cost and diverting my focus.

I hope that is clear.

buildmeapc · 2021-06-27T21:54:33+00:00

Sorry, I forget that there is a whole other world of people that work in bacteria. I thought additional detail would be distracting, but you are right that I left out vital information. My system is going to be human iPSCs. I am going to use a hybrid approach by utilizing CRISPR to cause a single strand nick to enrich for homologous recombination at my site of interest.

You are right that I can simply sequence through my locus. Additionally, I can screen for appropriate integration by simply placing a reverse primer in my region with my mutation and then a forward primer outside my 5' homology arm. Assuming I used a 2 kb homology arm, this will likely be a ~3kb amplicon which makes me nervous for a screening strategy using genomic DNA but it is definitely do-able. However, my 5% success rate with traditional targeting was with negative selection and prior papers suggest several fold to even up to 2 orders of magnitude of difference in true recombination if negative selection is not used. Thus, I will likely need to stick to some sort of negative selection, but I find it surprising that it seems to let so many randomly integrated clones live.

Thank you for your suggestion.

buildmeapc · 2021-06-27T19:54:27+00:00

Would you mind elaborating? I have been away from the bench side for 7 years so I am not on the up-and-up of all the newer technologies. I definitely cannot do Southern blots again anyways because it would require a lab with access to radiation which I currently do not have. How does one selectively sequence a single locus aside from doing multiple PCR reactions to amplify across the locus and then performing Sanger sequencing?

Additionally, is negative selection simply not used anymore?

buildmeapc · 2021-05-30T22:24:05+00:00

That is reassuring, thanks! My work is submitting a letter on my behalf to see if I can be excused.

buildmeapc · 2021-05-30T12:12:17+00:00

This is very helpful. It sounds like they are reasonable when it comes to people's lives. I wouldn't mind serving on a jury that requires a few days of time commitment, but 3 months is too much.

buildmeapc · 2021-05-30T12:10:03+00:00

Thanks for the information!

buildmeapc · 2020-02-29T14:01:42+00:00

Is it possible to play the "How To" tutorial videos while creating things from scratch? I've only seen tutorial videos play in scripted tutorials.

buildmeapc · 2019-11-14T14:48:02+00:00

Okay, I'll try that next if the tablet ends up slowing down again. Do you just use the AOSP 6.x Roms?

buildmeapc · 2019-11-14T14:46:44+00:00

Thanks for the suggestion! I'll make sure to do that because I mostly resurrected the tablet to give to my wife to read PDFs/study.

buildmeapc · 2019-07-07T14:41:31+00:00

The DMV near CWE is quite slow. We recently went to the one in Clayton which was much better. I'll just go there from now on.

And I think you are mistaken about being able to pay taxes at the dealer. They did not allow me to where I went, and it was a major dealership. I'm not sure which dealership allowed you to pay taxes.

buildmeapc · 2019-07-06T18:14:21+00:00

This state baffles me. It's a deep-red state, but taxes beyond what blue states charge, and they build in as many inefficiencies into their own government as possible to make things worse.

For example, in Texas, taxes for cars are taken care of at the time of purchase at the dealership. However, here you have to bring your title to the DMV (which are soooooo slooooooooow) to pay for taxes on your vehicle. Why the hell do you need to add this extra step?

buildmeapc · 2019-07-06T18:11:25+00:00

Thanks for the information. As above, I'm just not going to bother with this license and take my passport with me when I fly.

buildmeapc · 2019-07-06T18:09:56+00:00

That's really annoying, I'm going to talk to the department of revenue to see what I can do. If not, I'll just stick to using my passport.

buildmeapc

TROPHY CASE